Apr 24, 2026
Ventral midbrain dopaminergic (mDA) Neuron Monolayer Differentiation Protocol
- 1National Human Genome Research Institute
Protocol Citation: Yu Chen 2026. Ventral midbrain dopaminergic (mDA) Neuron Monolayer Differentiation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrw28zlmk/v1
Manuscript citation:
Chase Chen, Charis Ma, Richard Sam, Jens Lichtenberg, Tiffany Chen, Ying Hao, Ziyi Li, Isabelle Kowal, Kate Andersh, Yue Andy Qi, Gani Perez, Ellen Hertz, Yan Li, Darian Williams, Mark J. Henderson, Morgan Park, Xuntian Jiang, Pilar Alvarez Jerez, Cornelis Blauwendraat, Ellen Sidransky, Yu Chen
bioRxiv 2025.06.23.661126; doi: https://doi.org/10.1101/2025.06.23.661126
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 09, 2025
Last Modified: April 24, 2026
Protocol Integer ID: 229434
Keywords: ASAPCRN, iPSC, midbrain dopaminergic neurons (mDA), Parkinson's disease, Differentiation, ASAPCRN, optimizations for ht809 gba1 isogenic ipsc line, neuron monolayer differentiation protocol generating, ventral midbrain dopaminergic neuron, chir boost phase, dopaminergic neuron, ventral midbrain dopaminergic, ht809 gba1 isogenic ipsc line, concentration of chir999021, induced pluripotent stem cell, pluripotent stem cell, vulnerable in parkinson, parkinson
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000472
Abstract
Generating authentic functional ventral midbrain dopaminergic neurons, those most vulnerable in Parkinson's disease, from human induced pluripotent stem cells (iPSCs) has enabled cell replacement therapy clinical trials and increasingly sophisticated in vitro disease models. This protocol is our optimizations for HT809 GBA1 isogenic iPSC lines on a recently developmental patterning approach (Kim et al., 2021), focusing on optimizing the concentration of CHIR999021 for the CHIR Boost phase.
Guidelines
CHIR Dose:
CHIR99021 concentration may have to be altered to obtain the highest mDA neuron differentiation efficiency for different iPSC / ESC cell lines due to line-intrinsic differences in Wnt signaling tone and interacting signalling pathways.
Materials
Accutase, ThermoFisher Scientific, A1110501
Geltrex, ThermoFisher Scientific, A1413302
Neurobasal Medium, ThermoFisher Scientific, 21103-049
GlutaMAX, ThermoFisher Scientific, 35050079
N2 supplement, ThermoFisher Scientific, 17502048
B-27 supplement without Vitamin A, ThermoFisher Scientific, 12587010
Culture CEPT Supplement (1000X) A56799
Laminin-521, ThermoFisher Scientific, A29249
LDN 193189 dihydrochloride, TOCRIS, Cat# 6053
A 83-01, TOCRIS, Cat# 2939
SAG 21k, TOCRIS, Cat# 5282
human Sonic Hedgehog/Shh (C24II) N-Terminus, R&D systems, 1845-SH
CHIR99021, TOCRIS, Cat# 4423
Dibutyryl cAMP sodium salt, Millipore Sigma, Cat# D0627
DAPT, TOCRIS, Cat# 2634
Transforming growth factor type β3 (TGFβ3), R&D Systems, Cat# 243-B3
Brain-derived neurotrophic factor (BDNF), Peprotech, 450-02
Glial cell line-derived neurotrophic factor (GDNF), Peprotech, 450-10
Ascorbic acid, Sigma-Aldrich, 4034
Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract, R&D Systems, Catalog #: 3434-010-02
Safety warnings
CHIR99021 concentrations above 7.5 μM results in dose-dependent cell death ( Kim et al., 2021)
iPSCs Culture
iPSCs were cultured using Essential 8 (E8) media on Vitronectin (VTN-N)-coated plates. For passage, iPSCs were incubated with 0.5 mM EDTA for 00:05:00 at 37 °C . After EDTA was removed, iPSCs were dissociated in E8 media + CEPT (or Y27632) and seeded into VTN-N-coated plates.
Neural Induction and Patterning
Day 0: iPSCs seeding in Neural Induction Media (NIM) on Geltrex-coated plates
Coat plates with Geltrex
Geltrex was diluted in DMEM/F12 media at 1:100 to coate plates. 1 mL is used for each well of a 6-well plate. Plates need be coated at 37 °C for at least 01:00:00
Prepare NIM + CEPT. 4 mL is needed to seed 4 million iPSCs in one well of a 6-well plate.
NIM
| Components | Working concentration | For 500 mL | |
| Neurobasal | 500 mL | ||
| N2 supplement | 5 mL | ||
| B27 supplement w/o Vitamin A | 10 mL | ||
| Glutamax | 5 mL | ||
| LDN193189 | 250 nM | ||
| A83-01 | 2 uM | ||
| SHH-C24II | 250 ng/mL | ||
| SAG 21k | 2 uM | ||
| CHIR99021 | 0.75 uM |
Dissociate iPSCs with Accutase and seed in NIM + CEPT on Geltrex-coated plates at 400k cells/cm2.
Day 1, 2, 3: Daily feeding with NIM.
Complete medium change is conducted daily with 4 mL NIM for each well of a 6-well plate.
Day 4, 5, 6, 7: Daily feeding with NIM + CHIR boost
Complete medium change is conducted daily with 4 mL NIM + CHIR boost for each well of a 6-well plate.
NIM + CHIR boost
| Components | Working concentration | For 500 mL | |
| Neurobasal | 500 mL | ||
| N2 supplement | 5 mL | ||
| B27 supplement w/o Vitamin A | 10 mL | ||
| Glutamax | 5 mL | ||
| LDN193189 | 250 nM | ||
| A83-01 | 2 uM | ||
| SHH-C24II | 250 ng/mL | ||
| SAG 21k | 2 uM | ||
| CHIR99021 | 3 uM |
Day 8, 9: CHIR Boost
Complete medium change is conducted daily with 4 mL CHIR boost for each well of a 6-well plate.
CHIR boost
| Components | Working concentration | For 500 mL | |
| Neurobasal | 500 mL | ||
| N2 supplement | 5 mL | ||
| B27 supplement w/o Vitamin A | 10 mL | ||
| Glutamax | 5 mL | ||
| CHIR99021 | 3 uM |
Day 10: Switch to Neural Patterning Media (NPM) + CHIR Boost
Complete medium change is conducted with 4 mL NPM for each well of a 6-well plate.
NPM + CHIR Boost
| Components | Working concentration | For 500 mL | |
| Neurobasal | 500 mL | ||
| B27 supplement w/o Vitamin A | 10 mL | ||
| Glutamax | 5 mL | ||
| BDNF | 20 ng/mL | ||
| GDNF | 20 ng/mL | ||
| Ascorbic Acid | 200 uM | ||
| db-cAMP | 200 uM | ||
| TGFb3 | 1 ng/mL | ||
| CHIR99021 | 3 uM |
Day 11: Plating mDAN progenitors at 800k cels /cm2 in NPM + CHIR Boost + CEPT on Geltrex-coated plates.
mDAN progenitors were dissociated with Accutase by incubating 00:10:00 at 37 °C .
mDAN progenitors were resuspended in NPM + CHIR Boost + CEPT and seeded at 800k cels /cm2 on Geltrex-coated plates.
Day 12: Feed with NPM + CHIR Boost.
Complete medium change is conducted with 4 mL NPM + CHIR Boost for each well of a 6-well plate.
Day 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24: daily feeding with NPM
Complete medium change is conducted with 4 mL NPM for each well of a 6-well plate.
NPM
| Components | Working concentration | For 500 mL | |
| Neurobasal | 500 mL | ||
| B27 supplement w/o Vitamin A | 10 mL | ||
| Glutamax | 5 mL | ||
| BDNF | 20 ng/mL | ||
| GDNF | 20 ng/mL | ||
| Ascorbic Acid | 200 uM | ||
| db-cAMP | 200 uM | ||
| TGFb3 | 1 ng/mL | ||
| DAPT | 10 uM |
Note: mDAN progenitors can be frozen down at day 16 in STEM-CELLBANKER at 10 to 30 million per mL.
Day 25: mDAN progenitor seeding for maturation.
Coat plates with Cultrex
Cultrex was diluted in DMEM/F12 media at 1:100 to coate plates. 1 mL is used for each well of a 6-well plate. Plates need be coated at 37 °C for at least 01:00:00
Note: DANs attach to Cultrex better than to Geltrex.
Plating mDAN progenitors at 50k~200k cells /cm2 in NPM + CEPT on Cultrex-coated plates.
mDAN progenitors were dissociated with Accutase by incubating 00:10:00 at 37 °C .
mDAN progenitors were resuspended in NPM CEPT and seeded on Cultrex-coated plates.
NPM for maturation
| Components | Working concentration | For 500 mL | |
| Neurobasal | 500 mL | ||
| B27 supplement w/o Vitamin A | 10 mL | ||
| Glutamax | 5 mL | ||
| BDNF | 20 ng/mL | ||
| GDNF | 20 ng/mL | ||
| Ascorbic Acid | 200 uM | ||
| db-cAMP | 500 uM | ||
| TGFb3 | 1 ng/mL | ||
| DAPT | 10 uM |
Neural Maturation
Day 25 onward: feeding every 72 hours with NPM for maturation.
NPM for maturation
| Components | Working concentration | For 500 mL | |
| Neurobasal | 500 mL | ||
| B27 supplement w/o Vitamin A | 10 mL | ||
| Glutamax | 5 mL | ||
| BDNF | 20 ng/mL | ||
| GDNF | 20 ng/mL | ||
| Ascorbic Acid | 200 uM | ||
| db-cAMP | 500 uM | ||
| TGFb3 | 1 ng/mL | ||
| DAPT | 10 uM |
Protocol references
Kim TW, Piao J, Koo SY, Kriks S, Chung SY, Betel D, Socci ND, Choi SJ, Zabierowski S, Dubose BN, Hill EJ, Mosharov EV, Irion S, Tomishima MJ, Tabar V, Studer L. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. Cell Stem Cell. 2021 Feb 4;28(2):343-355.e5. doi: 10.1016/j.stem.2021.01.005. PMID: 33545081; PMCID: PMC8006469